Production and characterisation of novel recombinant antibody fragments against sialic acid

Sialic acids are a family of acidic monosaccharides that typically reside as terminal moieties on N- and O-linked glycans. These sugars are actively involved in a plethora of biological phenomena, ranging from cell-cell adhesion and recognition, intracellular signalling events, pathogen attack, viral infection and inflammatory disease. In addition, many cancer-related antigens contain terminal sialic acids or altered sialylation patterns. The identification of sialic acid-specific antibodies is important in the fields of basic research and diagnostics. Therefore the primary objective of this thesis was the generation and characterisation of recombinant antisialic acid antibodies.A single-chain antibody fragment (scFv) library was constructed from a chicken that was immunised with a novel synthetic sialic acid protein-conjugate (Neu5Gchumanserum albumin). The scFv library was biopanned using a second novel sialoneoglycoconjugate(Neu5Gc-bovine serum albumin). Anti-sialic scFvs were isolated byphage-display and binding activity, multimeric status, and multivalent properties wereassessed by ELISA, HPLC, FPLC, and SPR. One clone, AE8, displayed the strongest reactivity to a panel of different sialylated structures. In addition, SPR inhibition andkinetic analysis revealed that the AE8 scFv had nanomolar affinity for the Neu5Gc-BSA neoglycoconjugate. The heavy chain gene of the AE8 scFv was assembled with a repertoire of error prone PCR-amplified light chain genes, that originated from the unpanned anti-sialic acid library. The mutant scFv library was biopanned using a depletion protocol and two ovalbumin (OVA) conjugates, Neu5Gc-linker1-OVA and linker1-OVA. ELISA analysis, SPR inhibition and ‘off-rate’ studies identified one mutant clone, E15, which displayed strong affinity for conjugates of sialic acid. A solution-phase SPR-based inhibition study demonstrated that the E15 scFv had an IC50 concentration of 1.6ng/mL for the Neu5Gc-BSA conjugate. When compared to the parental AE8 clone (IC50 of 5.7ng/mL) the mutant E15 scFv exhibited an improvement in sialic acid binding of approximately 3.5-fold. The generation of such highly specific anti-sialic acid antibodies in a recombinant format is exceedingly rare. Most anti-carbohydrate antibodies are not recombinant and display weak carbohydrate-protein interactions. This work shows for the first time, the generation and directed evolution of anti-carbohydrate scFvs that have nanomolar affinity for Neu5Gc-containing structures.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Dublin City University

Year dc:date.issued

2011

Author dc:creator

• Donohoe, Gerard Gary

• Biotechnology

Language dc:language

en